Region specific remodelling of synaptic contacts in the dentate nucleus and in the pontine nuclei

We found equal synaptophysin signal density in chronic cortical MS plaques and in cortex of non-MS control brains. This result suggests either preserved density of presynaptic boutons or reappeared boutons reinnervating the preserved postsynaptic partner neurons in the lesions.

Furthermore, we extended our observation that frontal cortex is often involved in chronic MS by showing that remodelling of synaptic contacts may take place in motor relay stations, such as the dentate nucleus and the pontine nuclei. Rather than being induced by local (humoral) factors, MS-related forms of synaptic remodelling may – at least in part - be based on transsynaptic and transneuronal effects of focal demyelination (Blinzinger and Kreutzberg, 1968). The structural changes observed suggest that at least two different mechanisms may be involved: (i) the so-called “synaptic stripping” (Blinzinger and Kreutzberg, 1968), that has been shown to be selective and reversible during ontogenetic development of spinal motorneurons (Conradi and Ronnevi, 1975) and as a retrograde reaction to experimental injury of axons (Moran and Graeber, 2004; Olmos et al.; 1989) and (ii) autophagy and lysosomal degradation of synaptic elements (Wolff et al. 1981; Gallyas et al., 1980) and subsequent transport of residual bodies in intact axons.

Discussion 4.4.1. Region specific synaptic remodelling in the dentate nucleus

Our immunhistochemical studies showed that the characteristic axo-somatic innervation pattern was eliminated selectively in patients with chronic MS; i.e. the population of large GAD-positive boutons forming axo-somatic synapses were lost from a subpopulation of neurons, while GABAergic and non-GABAergic innervation of their dendrites was apparently preserved. These observations indicate that in MS patients, neurons of the dentate nucleus may selectively lose input from the inhibitory feedback-loop provided by Purkinje cell axons.

Other synapses are apparently preserved, especially those that are located on peripheral dendrites and are preferentially composed of small GABAergic and non-GABAergic boutons.

The conditions, which might induce axo-somatic denervation remain obscure. The fact that we haven’t found cell death of Purkinje cells (not shown) fits to the concept that axo-somatic synapses in the dentate nucleus did not undergo anterograde (Wallerian) degeneration. Also, demyelination of the respective Purkinje cell axons is probably not the reason. Although dissociation of axo-somatic synapses (synaptic stripping) was regularly found in demyelinated parts of the dentate nucleus, it also occurred in periplaque regions and – most importantly - also developed in cases without recognizable demyelination in the cerebellum. Thus, synaptic regression in the dentate nucleus might be induced by other conditions, which are determined by the disease itself. Humoral agents are not very likely inducers, because adjacent neurons would react differently. However, suspension of the cerebellar function is more likely.

Alterations in connectivity may take place due to damage to any part of the cortico-ponto-cerebellar tracts. This may explain the consistent reduction of axo-somatic innervation in the dentate nucleus of every MS patients studied. On the other hand, many MS patients are immobilized for long periods of time, i.e. vestibular stimuli and cerebellar compensatory functions are reduced to a minimum. Unfortunately, there are no comparable studies available on effects of long-term immobilisation on cerebellar nuclei.

4.4.2. Displacement of synapses upon postsynaptic induction in the dentate nucleus: synaptic stripping

Four modes of synaptic regression have been reported so far. Two of them occur as an irreversible consequence of cell death of pre- and/or postsynaptic neurons and/or degeneration of cell processes. The other two mechanisms, i.e. synaptic stripping and lysosomal degradation of predominantly the presynaptic elements lead to reversible disconnection of synaptic junctions (Wolff et al.; 1995). By electron microscopy, we showed dissociation of the synaptic boutons from the soma membrane by processes of glial cells in the dentate

Discussion nucleus. Dissociation of the pre- and post-synaptic structures is accompanied by several

structural changes, such as local impairment of membrane adhesion, expansion of the intercellular space and insulation of the soma membrane with stacks of compacted astrocytic lamellae. This way of synaptic regression is reminiscent of the process that has been initially described on the soma of developing motoneurons (Conradi and Ronnevi, 1975) and experimentally induced by axotomy (Blinzinger and Kreutzberg, 1968). According to the latter authors, facial nerve transection leads to proliferation and activation of microglial cells near the cell bodies of motorneurons in the facial nucleus. Microglia cells seem to be involved in separating the axonal boutons from the soma membrane of the regenerating motoneurons.

Intercellular clefts dilate following activation of extracellular proteases (e.g. tissue plasminogen activator) in this way providing space for astrocytic processes that take over the perineuronal positions previously occupied by presynaptic elements of axo-somatic synapses and microglial cells. Interestingly, the disconnection of axo-somatic synapses is selective, i.e.

it is restricted to excitatory synapses on motorneurons and reversible when neuronal function in circuits is restituted. This mode of synaptic disconnection is not confined to pathological conditions, but may occur during development of spinal motoneurons (Conradi and Ronnevi, 1975) as well as on pyramidal neurons in the developing cerebral cortex (Bähr and Wolff, 1985). Synaptic stripping was also shown in the arcuate nucleus, were detachment and reappearance of the axo-somatic boutons select for GABA-ergic synapses. Nevertheless, conditions responsible for the synaptic stripping are obviously different. In the arcuate nucleus it is inducible by oestrogen in a concentration-dependent manner (Cardona-Gomez et al., 2000; Naftolin et al., 1996; Parducz et al., 1993; Olmos et al.; 1989), while in the dentate nucleus humoral induction is improbable (see above). Considerable differences that exist among various experimental models do not help in answering the question, how the detachment of boutons from soma membranes is induced in the dentate nucleus in MS. If it occurred ‘on postsynaptic demand’ (as it seems to be the case in a retrograde axotomy reaction) projections from the dentate nucleus to the thalamus might be disturbed.

Unfortunately, we did not have the necessary tissue material available to describe synaptic contacts in ventrolateral thalamic nuclei to examine this possibility.

4.4.3. Displacement of synapses upon presynaptic induction in the dentate nucleus:

autophagy and lysosomal degradation of synaptic components

The fourth and also reversible way of structural modifications of synaptic junctions is autophagy and lysosomal degradation of predominantly presynaptic elements (Wolff et al.;

Discussion 1989; Wolff et al.; 1981; Gallyas et al., 1980). We showed in the demyelinated dentate

nucleus the selective fusion and autophagy of synaptic vesicles surrounded by voluminous astroglial processes that contained glycogen grains and bundles of astroglial intermediate filaments (GFAP-positive structures). Lysosomal degradation may be induced by invagination of parts of presynaptic elements, spinules from the postsynaptic dendrite or finger-like processes from astrocytes. This stage may be characterised by astrocytic swelling and increase in S100-immunreactivity. It is followed by the formation of autophagosomes including synaptic vesicles or mitochondria apparently leading to reduction in synaptic function. During this regressive process the surrounding glia is frequently characterised by increased GFAP immunreactivity and pools of glycogen grains (Wolff et al.; 1995). Lysosomal degradation may either reduce the transmitter content or the capacity of release by removing synaptic vesicles and/or parts of the presynaptic specialisations. It may lead to empty presynaptic elements, i.e. the transmitter release may be completely suspended. These changes are reversible as long as the interneuronal connection persists. However, lysosomal degradation may also include postsynaptic structures and, thus, can result in disconnection and removal of both, pre- and postsynaptic densities, thus elimination of whole synapses (Wolff et al.; 1995).

4.4.4. Preserved synaptic density in the demyelinated pons

Synaptic density was histologically characterised by the number of synaptophysin-immunoreactive puncta per area of tissue section. This was largely unaltered in the demyelinated zones of pons, though some structural changes were observed. Occasionally, rarefied and/or focally aggregated synaptophysin-positive material indicated that synaptic vesicles might be redistributed in subpopulations of axonal boutons. Such changes were not only seen in demyelinated plaques. It was also seen in periplaque zones and in the contralateral parts of the respective nucleus but not in the control cases. Thus, we did not find any consistent histological change that could be attributed to demyelination in the pontine nuclei. Nevertheless, it is possible that the reduced synaptic density and focal accumulation of synaptic signals in the nuclei contralateral to the lesion resulted from transsynaptic changes mediated by communicating fibres that are crossing the midline of the pons.

4.4.5. Multicellular dynamic synaptic reorganisation in the demyelinated pons

Despite the scarce histological findings, electron microscopy provided evidence for structural changes by which synaptic remodelling could be identified in the demyelinated pons.

Numerous synaptic boutons contained large aggregates of vesicles that were dislocated from the active zones of the synaptic junctions. Such vesicle aggregates probably can not

Discussion participate in synaptic transmission and reduce the probability of the quantal release in

response to action potentials. In addition, numerous free/vacated postsynaptic densities were found in the demyelinated pons. Such vacant postsynaptic densities may either be formed, when pre-and postsynaptic elements of a synapse have been dissociated from each other or the presynaptic element underwent regression while the postsynaptic one persisted (synaptic regression ‘upon presynaptic demand'). Alternatively, there is postsynaptic demand for formation of new synapses; and in this latter case the free/vacant paramembraneous densities may serve as target sites for the formation of new synapses, when compatible presynaptic offers are available. Such a situation occurs during ontogenesis, when formation of synaptic contacts depends on the spatio-temporal matching of the potential pre- and postsynaptic elements. Pre- and postsynaptic elements, however, may be formed independently or each in different numbers. In this case, vacant postsynaptic densities appear and axon varicosities are found without synaptic contacts (Wolff et al. 1995; Dammasch et al., 1986; Wolff and Wagner 1983). Astrocytes play an active role to match the pre- and the postsynaptic contact offerings forming new intercellular connections. From static electron microscopic pictures one can thus suspect that synaptic reorganisation is going on in the pons in MS, which is orchestrated and accompanied by astroglial cells, as suggested (according to the Tripartite-Synapse Concept) by Haydon et al. (2001). Correspondingly, voluminous processes of astrocytes appeared nearby axonal varicosities and opposite to free/vacant paramembranous densities. On the one hand, intermittent astrocytes may prevent reinnervation on adjacent vacant postsynaptic densities but on the other hand, astrocytes match axonal varicosities and free postsynaptic densities and assist in forming new synaptic relationships via connecting the pre- and postsynaptic partner neurons. The presence of abundant primary lysosomes (e.g.

multivesicular bodies) in pre- and postsynaptic elements and in the perisynaptic astroglia, however, indicate that all three partners may be involved in regulating synaptic reorganisation upon the functional demand of the neuronal network.

Summary 5. SUMMARY

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. MS has traditionally been considered a disease affecting the well myelinated white matter areas. Recent insights into axonal pathology in MS raised the attention to the significance of grey matter lesions, which have not been well characterised yet. Thus, the aim of our study was to delineate the fundamental histopathological aspects of MS lesions in the grey matter.

Our work identified extensive cortical demyelination associated with marginal inflammation in autopsy brain tissue of patients with chronic MS. In addition, we found evidence for T-cell/macrophage-mediated active inflammatory demyelination in cortex in biopsy brain tissue of patients with early MS. A direct comparison of the inflammatory activity in white matter and cortical lesions of the same patients revealed that the density and composition of T-lymphocytes was similar in early grey and white matter lesions. Foamy macrophages were, however, essentially absent; the blood brain barrier appeared intact, acute axonal injury was less in the cortical in contrast to the white matter lesions. While acute neuronal injury was apparent in a proportion of early lesions, neurons with synaptic boutons were well preserved in the chronic grey matter plaques.

We examined the frequency and extent of remyelination in cortical and white matter lesions of patients with chronic MS. Cortical remyelination was identified light microscopically by the presence of irregularly arranged and less densely packed myelin sheaths, and confirmed by electron microscopy. A direct comparison of the extent of remyelination in white matter and cortical lesions of the same patients revealed that remyelination of cortical lesions was consistently more extensive. In addition, g-ratios of fibers in the “normal appearing cortex”

yielded values consistent with remyelination.

Preserved neuronal cell bodies and synaptic boutons as well as the extensive remyelination in chronic MS cortex suggested efficient repair and adaptive mechanisms that may take place in the grey matter during the course of MS. Therefore, the density, topography and morphology of synapses were investigated in the cerebellar dentate nucleus and in the nuclei of the pons by light and electron microscopy. There was a substantial loss of axosomatic synaptic contacts in the dentate nucleus in all MS patients examined. Synapses on the stem and peripheral dendrites, however, appeared well preserved. Subpopulations of neurons were affected to a variable degree. Dissociation of boutons from the soma membrane occurred

Summary irrespective of the lesional border; moreover, it could be found in sections, where no

demyelinated lesions were recognised. In the pontine nuclei the density of synapses appeared largely preserved. The structural changes observed suggested the mechanisms that may be involved: (i) “synaptic stripping” that has been shown in various experimental models to be selective and reversible, (ii) autophagy and lysosomal degradation of synaptic elements and subsequent transport of residual bodies in intact axons, which has been shown during ontogenetic development to be a mode of reorganisation of synaptic contacts.

Our data imply that inflammatory demyelination occurs in cortex even in early MS. Cortical de- and remyelination are frequent and the propensity to remyelinate is high in cortical MS lesions. Remodelling of synaptic contacts may take place even in late stages of the disease.

Further studies are required to determine the conditions under which regeneration can be elicited and supported, thus providing functional improvement of patients suffering from MS.

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